Method of disulfide crosslink forming in vitro protein synthesis

ABSTRACT

It is an objective of the present invention to provide a method of synthesizing a protein having disulfide bonds using a reconstituted protein synthesizing system in a convenient manner with high efficiency. It has been found that a protein having the activity can be obtained and synthesized with good efficiency by using a reconstituted protein synthesizing system comprising purified components, from which enzymes and substrates influencing an oxidation-reduction state are removed and in which a state of redox equilibrium between disulfide and thiol is artificially regulated. Such system is used instead of a cell extract or a crude fraction thereof that contains various enzymes and substrates that maintain a reduced state, such as thioredoxin reductase [EC 1.6.4.5] and glutathion reductase [EC 1.6.4.2], resulting in difficulty in conditioning a redox state.

TECHNICAL FIELD

The present invention relates to a protein synthesis method using an in vitro transcription/translation system. More specifically, the present invention relates to a method of synthesizing a protein having intermolecular/intramolecular disulfide bonds using an in vitro transcription/translation system with efficiency.

BACKGROUND ART

It has been widely attempted to produce a protein that can be used as a pharmaceutical and a reagent via gene recombination technology. Preferably, in view of the ease of handling and efficiency, microorganisms such as E. coli, Bacillus subtilis, molds, and yeasts, insects such as silkworms, mammalian animals such as bovines, and culturable plant, insect, and animal cells have recently been used in gene recombination technology. A method of producing a protein using gene recombination technology has been widely used. However, the use of such method results in, for example, a low expression level for a protein of interest, the expression of a proteins having no activity, and formation of aggregates, which have been problematic. Thus, it is necessary to carry out a trial-and-error process such as examination of culture conditions, growth conditions, or induction conditions, or testing of various types of expression systems. Even after examination of these various conditions, many proteins that are difficult to produce have been reported.

Meanwhile, a protein synthesis method that does not use any such organisms or cells, called “cell-free protein synthesis,” has been known. Such cell-free protein synthesizing system is also described as an “in vitro transcription/translation system.” With this system, a template gene is subjected to transcription/translation using an extract or a crude fraction thereof prepared from E. coli, rabbit reticulocytes, wheat germ cells, or the like, resulting in protein synthesis. A cell-free protein synthesizing system is characterized in that limitations associated with the use of organisms and cells can be overcome. This is because, with the use of such system, there is a high probability that a protein that disturbs functions of organisms and cells can be synthesized, various types of proteins can be synthesized using a 96-well or 384-well format, and a variety of synthesis reaction conditions can be simultaneously examined.

However, it has been known that, even with the use of such cell-free protein synthesizing system, synthesized proteins form aggregates and do not constitute the relevant original structures, which have been problematic. For that reason, it is considered that proteins having intramolecular and/or intermolecular disulfide bonds are unable to properly crosslink between disulfide bonds. There are proteins having intramolecular and/or intermolecular disulfide bonds and those not having the same. Many of proteins that are transported and secreted onto the cellular surface or into the extracellular environment have disulfide bonds. Such proteins are believed to have particularly useful application. For instance, most of protein preparations such as insulin, cytokine, and blood cell growth factors that have been commercially available have a protein having intramolecular disulfide bonds as a component.

Thus far, in order to produce such proteins having intramolecular/intermolecular disulfide bonds with good efficiency, cell-free protein synthesizing systems have been improved. For instance, the following methods have been carried out: a method of adding microsome fractions to a cell extract (Non-Patent Document 1: Biochem. J. 254:805-810 (1988), hereafter referred to as Prior art 1); and a method of dialyzing a cell extract, a method of adding oxidized glutathione and reduced glutathione, a method of gel filtration, or a method of regulating oxidation-reduction (redox) potential (Non-Patent Document 2: FEBS Lett. 514:290-4 (2002); Non-Patent Document 3: Nature Biotech. 15:79-84 (1997); Patent Document 1: JP Patent Publication (Kokai) No. 2003-116590; Patent Document 2: WO 03/072796 A1, hereafter referred to as Prior art 2).

Also, a method of disulfide bond formation using a deletion variant of an enzyme that maintains a reduced state has been known (Proc. Natl. Acad. Sci. 96: 13703-13708 (1999), hereafter referred to as Prior art 3). With this method, a protein of interest can be expressed using E. coli lacking thioredoxin reductase and glutathion reductase. However, in general, such an enzyme-deficient cell line grows very slowly or requires specific culture conditions, which have been significant obstacles in terms of industrial availability. Such method employs a system that causes gene recombination to be carried out in E. coli such that proteins are expressed. However, as described above, there have been various limitations associated with the direct use of organisms. In addition, many organisms contain many unidentified enzymes and substrates controlling oxidation-reduction in addition to thioredoxin reductase and glutathion reductase (Non-Patent Document 4: Nature Review Molecular Cell Biology 3: 836-847 (2002)). Thus, even if a foreign gene can be expressed using such a deletion variant or protein synthesis can be carried out in a cell-free protein synthesizing system using a cell extract of a deletion variant, stable disulfide bond formation has been considered to be difficult. Therefore, a method of treating a cell extract with iodacetamide so as to inactivate such enzymes and substrates has been known (U.S. Pat. No. 6,548,276 B2, hereafter referred to as Prior art 4). In accordance with this method, it is possible to inactivate not only glutathion reductase and thioredoxin reductase but also many enzymes and substrates regulating intracellular oxidation-reduction. However, since iodacetamide modifies thiol in a nonspecific manner, it also modifies factors and enzyme groups related to transcription/translation, ribosomal protein, and the like. It is considered that efficiency or accuracy of an in vitro protein synthesis reaction deteriorates as a result, which is problematic.

Meanwhile, in accordance with a method that has been widely used, proteins obtained via recombinant production using organisms and cells, chemically synthesized peptides, or proteins synthesized via a cell-free protein synthesizing system are completely denatured using a denaturant, followed by regeneration thereof (Non-Patent Document 5: Biochemistry 26:3129-3134 (1987), hereafter referred to as Prior art 5). In the case of Prior art 5, since proteins are chemically synthesized or recovered as inactive insoluble matter when using organisms, toxic proteins can be produced, for example. Upon protein regeneration, cells are not used and reagents and salts freely combine with each other so that a proper disulfide bond can be introduced into a protein having disulfide bonds. When carrying out this method, however, separate steps of protein synthesis and protein structure regeneration are required, and protein structure regeneration is a time-consuming step (taking several days to one week), which have been problematic.

Further, in accordance with a method of measuring activity of an enzyme catalyzing promotion and/or isomerization of disulfide bonds that has been widely known, ribonuclease A (RNaseA) serving as a substrate is reduced so as to be denatured, RNaseA is refolded together with an enzyme, the activity of which is measured, so as to be regenerated, and the activity of RNase obtained is determined to be an index (Non-Patent Document 6: Biochem J. 1976 159:377-384).

-   Patent Document 1: JP Patent Publication (Kokai) No. 2003-116590 A -   Patent Document 2: WO 03/072796 A1 -   Patent Document 3: U.S. Pat. No. 6,548,276 B2 -   Non-Patent Document 1: Biochem. J. 254:805-810 (1988) -   Non-Patent Document 2: FEBS Lett. 514:290-294 (2002) -   Non-Patent Document 3: Nature Biotech. 15:79-84 (1997) -   Non-Patent Document 4: Nature Review Molecular Cell Biology     3:836-847 (2002) -   Non-Patent Document 5: Biochemistry 26:3129-3134 (1987) -   Non-Patent Document 6: Biochem J. 159:377-384 (1976)

DISCLOSURE OF THE INVENTION

[Protein Synthesis]

Conventional cell-free protein synthesizing systems have had the following drawbacks upon production of proteins having intramolecular and/or intermolecular disulfide bonds. In the case of Prior art 1, it is considered that: it is necessary to prepare microsome fractions from the pancreas or the like via homogenization and centrifugation; microsome fractions are obtained by disrupting organella called endoplasmic reticula in which intracellular disulfide bond formation originally takes place so that disulfide bond formation in microsome fractions is less efficient than that in endoplasmic reticula; and patterns of disulfide bonds formed differ from original patterns, so that the protein activity might not be observed, which have been problematic. In the case of Prior art 2, it is not necessary to prepare microsome fractions, and cell extracts can be prepared via a simple method such as addition of oxidized glutathione and reduced glutathione or dialysis. However, it has been difficult to prepare a reaction solution with good reproducibility of an oxidation-reduction state such that disulfide bonds can be crosslinked with good efficiency. This is because a cell extract that is usually used as a cell-free protein synthesizing system contains various types of enzymes and substrates maintaining a reduced state, including thioredoxin reductase [EC 1.6.4.5] and glutathion reductase [EC 1.6.4.2] (Nature Review Molecular Cell Biology 3:836-847 (2002)). Thus, it is considered that functions of such enzymes and substrates cause difficulties in conditioning an oxidation-reduction state, even with dialysis or addition of glutathione. When an oxidant such as oxidized glutathione (GSSG) is added to a cell-free protein synthesizing system, a redox buffer of GSSG and reduced glutathione (GSH) has been used. Usually, the ratio therebetween is about GSSG:GSH=1:5 to 1:10. This ratio is based on oxidant concentrations and ratios obtained from a variety of studies wherein RnaseA or lysozyme is reductively denatured for refolding so as to examine the correlation between structure and activity (Biochemistry 1991 30:613-619; Biochemistry 1970 9:5015-5023)). Thus, such ratio is not necessarily suitable for a cell-free protein synthesizing system. In addition, the term “oxidation-reduction potential” indicates potential derived from differences in the chemical potentials of an oxidized form and a reduced form, which is measured using an electrode such as a platinum electrode in an equilibrium state in which reversible electron transfer is achieved. Such potential is merely obtained based on the collective observation of potentials of various inorganic/organic substances (e.g., oxygen, metal, cysteine, and heme) in a redox equilibrium state, which are involved in oxidation-reduction in a solution. Thus, it is not an index showing a state of equilibrium between thiol and disulfide and thus it does not reflect a redox state of protein. Therefore, it has been significantly difficult to regulate disulfide bond formation in proteins even if an oxidation-reduction potential could be simply adjusted to a given level as shown in Prior art 2.

In both cases of Prior arts 1 and 2, protein synthesis efficiency itself might decrease, which is problematic. This is because, when a cell extract is subjected to treatments such as addition of microsomes, dialysis, or addition of oxidized glutathione and reduced glutathione, the intracellular environment becomes different from the original intracellular environment in which protein synthesis takes place. Thus, the use of a cell-free protein synthesizing system has been significantly difficult at an industrial level.

The inventors of the present invention have already disclosed a method wherein a part or all of protein components constituting a reaction system of a cell-free in vitro DNA transcription/translation system or RNA translation system are labeled with one of a pair of substances that adhere to each other (JP Patent Publication (Kokai) No. 2003-102495 A, hereafter referred to as reconstituted protein synthesizing system). This method completely differs from conventional cell-free systems and in vitro protein synthesizing systems (hereafter to be referred to as cell-free protein synthesizing systems). Specifically, the method is characterized in that different components involved in protein synthesis are purified without using a cell extract so as to be reconstituted such that protein synthesis is achieved, resulting in synthesis or rapid purification of proteins having no intramolecular disulfide bonds (Nature Biotechnology 19:732-734 (2001)). However, it has not been reported at what concentrations different components should be reconstituted in a manner such that proteins having intramolecular/intermolecular disulfide bonds can obtain their original structures and activities and can be synthesized with good efficiency.

Thus, a more convenient method of efficiently producing proteins having intramolecular and/or intermolecular disulfide bonds has been awaited. It is an objective of the present invention to provide a method of producing proteins having disulfide bonds using a reconstituted protein synthesizing system in a convenient manner with good efficiency.

[Enzyme Activity Measurement]

In accordance with the aforementioned conventional methods, it is required that RNaseA be temporarily denatured, and the step of refolding is time-consuming, which have been problematic.

[Protein Synthesis Method]

As a result of intensive studies in order to attain the above objective, the inventors of the present invention have found that a protein having intramolecular/intermolecular disulfide bonds and its original structure and activity can be obtained and synthesized with good efficiency by using a reconstituted protein synthesizing system from which enzymes and substrates influencing an oxidation-reduction state are removed and in which a state of redox equilibrium between disulfide and thiol is artificially regulated. This system is used instead of a cell extract or a crude fraction thereof that contains various enzymes and substrates that maintain a reduced state, such as thioredoxin reductase [EC 1.6.4.5] and glutathion reductase [EC 1.6.4.2], resulting in difficulty in conditioning a redox state.

Further, the inventors of the present invention have found that it is particularly preferable that a reconstituted protein synthesizing system be composed of ribosomes, initiation factors, elongation factors, termination factors, aminoacyl-tRNA synthetases, methionyl tRNA transformylases, tRNAs, amino acids, ribonucleoside triphosphates, 10-formyl 5,6,7,8-tetrahydrofolic acid (FD), salts, and water, which have purities of 90% or more. This has led to the completion of the present invention.

In general, at an intracellular protein synthesis site, a redox state is close to a reduced state, so that proteins synthesized in cells exist in a reduced state. In a cell-free protein synthesizing system, such intracellular state is reproduced so that DTT and other reducing reagents are generally contained in the system. However, it has been reported that, when reducing reagents are not contained, the quality of a preserved extract deteriorates or translational efficiency deteriorates (Eur. J. Biochem. 270:4780-4786 (2003)).

Meanwhile, in order to form disulfide bonds with thiol, a reduced state is not desirable. Thus, it has been attempted to remove DDT as a reductant from an in vitro transcription/translation system, or to decrease the amount of DTT added to such system for formation of disulfide bonds in proteins. However, as described above, the effects obtained have been limited.

On the other hand, in accordance with the method of the present invention, since the quantity of a component influencing a state of equilibrium between disulfide and thiol in a reconstituted protein synthesizing system is identified, DTT can be added in amounts of several μM to 1 mM. In addition, it is also possible not to add any DTT.

Moreover, in general, it has been considered that, in accordance with the conventional method using a cell-free protein synthesizing system from which DTT is removed, the amount of proteins synthesized decreases and the efficiency of synthesizing proteins of interest having activities deteriorates, even under a condition in which disulfide bonds can be formed in proteins. However, surprisingly, when a reconstituted protein synthesizing system is used as described in the present invention, the amount of proteins synthesized is not changed, or increases instead so that proteins can be synthesized with improved efficiency.

That is, in accordance with the present invention, unlike the conventional methods, a method of synthesizing proteins having disulfide bonds with high efficiency is provided, wherein: a protein synthesis reagent is reconstituted with ribosomes, initiation factors, elongation factors, termination factors, aminoacyl-tRNA synthetases, methionyl tRNA transformylases, tRNAs, amino acids, ribonucleoside triphosphates, 10-formyl 5,6,7,8-tetrahydrofolic acid (FD), salts, and water, which have been specified in terms of quantity and purity and contain no impurities influencing a redox state, without using a cell extract; and a state of redox equilibrium between disulfide and thiol is artificially conditioned in the reconstituted protein synthesizing system.

In addition, preferably, the aforementioned reconstituted protein synthesis reagent is reconstituted so as to contain predetermined amounts of ribosomes, initiation factors, elongation factors, termination factors, aminoacyl-tRNA synthetases, methionyl tRNA transformylases, tRNAs, amino acids, ribonucleoside triphosphates, 10-formyl 5,6,7,8-tetrahydrofolic acid (FD), salts, and water, which are highly purified and have purities of 90% or more. As an example of a protein synthesis reagent, a Pure system (Post Genome Institute Co., Ltd.) from which DTT has been removed can be used. In accordance with the aforementioned protein synthesis method, a state of redox equilibrium between disulfide and thiol can be artificially conditioned by adding (i) a reagent for conditioning a redox state and/or (ii) an enzyme catalyzing oxidation-reduction to the above protein synthesis reagent. Such addition may be carried out before the initiation of a reaction, during the reaction, or after the reaction.

More specifically, examples of (i) a reagent for conditioning a redox state include DTT and oxidized glutathione. Further, examples of (ii) an enzyme catalyzing oxidation-reduction include protein disulfide isomerases and disulfide interchange proteins.

In addition, the method of protein synthesis of the present invention may be referred to as a method of producing proteins.

[Method of Protein Activity Measurement and a Method of Protein Synthesis Using the Same]

Further, in accordance with the present invention, a test method is provided, comprising measurement of correlations among the activity of a protein produced, the amount of an oxidoreductase reagent catalyzing oxidation-reduction to be added, and the amount of a redox reagent catalyzing oxidation-reduction to be added. A protein synthesis reagent is reconstituted with predetermined amounts of ribosomes, initiation factors, elongation factors, termination factors, aminoacyl-tRNA synthetases, methionyl tRNA transformylases, tRNAs, amino acids, ribonucleoside triphosphates, 10-formyl 5,6,7,8-tetrahydrofolic acid (FD), salts, and water, which have been specified in terms of quantity and purity. Preferably, the concentrations of an oxidoreductase reagent and redox reagent that catalyze oxidation-reduction can be varied in the protein synthesis reagent. As described above, by adding reagents capable of changing a state of redox equilibrium to a reconstituted protein synthesizing system at desired final concentrations, it becomes possible either to form or not to form disulfide bond crosslinking in proteins produced.

That is, in accordance with the present invention, it becomes possible to properly know conditions required for producing proteins exhibiting desired crosslink formation of disulfide bonds using a reconstituted protein synthesizing system. The thus obtained information can be used with regard to the aforementioned protein synthesis conditions.

An oxidoreductase reagent and a redox reagent may be added before the initiation of a reaction, during the reaction, or after the reaction.

Examples of an oxidoreductase reagent include DTT and GSSG. Examples of a redox reagent include protein disulfide isomerases and disulfide interchange proteins.

Reagents at different concentrations can be prepared in advance so as to be added to respective reaction systems before the initiation of a reaction (before addition of a template nucleic acid), during the reaction, or after the termination of the reaction.

[Method of Enzyme Activity Measurement]

Specifically, a method of activity measurement may be carried out using the aforementioned protein synthesizing system in the manner described above except that an enzyme catalyzing promotion and/or isomerization of disulfide bonds, the activity of which is measured, is added upon or after translation. Proteins used as substrates are added as template DNAs or RNAs encoding such proteins. Types of proteins used as substrates are not particularly limited. Preferably, such proteins have known structures, and more preferably, they are enzymes. Examples of such substrates include lysozyme and alkaline phosphatase. In addition, in accordance with this method, the activity of an enzyme promoting disulfide bond formation and/or catalyzing disulfide bond isomerization can be measured, while on the other hand, a substance that inhibits the activity can also be screened for. In the case of screening, screening can be carried out using a test substance with an enzyme promoting disulfide bond formation or catalyzing disulfide bond isomerization.

[Kit]

Further, the present invention includes a kit comprising the following (1) a) and b), (2) a) and c), or (3) a), b), and c):

a) a protein synthesis reaction reagent comprising components that have been specified in terms of quantity and purity and causing a reaction to synthesize a protein encoded by a template nucleic acid upon addition of the template nucleic acid;

b) at least one oxidoreductase catalyzing oxidation-reduction, which has been specified in terms of quantity and purity; and

c) at least one redox reagent for conditioning a redox state, which has been specified in terms of quantity and purity.

Specifically, in accordance with the present invention, a kit comprising the following a) to c) is provided: a) protein synthesis reaction reagent comprising ribosomes, initiation factors, elongation factors, termination factors, aminoacyl-tRNA synthetases, methionyl tRNA transformylases, tRNAs, amino acids, ribonucleoside triphosphates, 10-formyl 5,6,7,8-tetrahydrofolic acid (FD), salts, and water, which have been specified in terms of quantity and purity and causing a reaction to synthesize a protein encoded by a template nucleic acid upon addition of the template nucleic acid; b) at least one oxidoreductase catalyzing oxidation-reduction, which has been specified in terms of quantity and purity; and c) at least one redox reagent for conditioning an oxidation-reduction state, which has been specified in terms of quantity and purity.

This description includes part or all of the contents as disclosed in the description of Japanese Patent Application No. 2004-136520, which is a priority document of the present application.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the office upon request and payment of the necessary fee.

FIG. 1 shows 6% to 36% sucrose density-gradient fractions of a ribosome crude extract.

FIG. 2 shows images of His-Tagged alanyl-tRNA synthetase (AlaRS), arginine tRNA synthetase (ArgRS), asparagine tRNA synthetase (AsnRS), aspartic acid tRNA synthetase (AspRS), cysteine tRNA synthetase (CysRS), glutamine tRNA synthetase (GInRS), and glutamic acid tRNA synthetase (GluRS) subjected to 12% SDS-PAGE/Coomassie brilliant blue staining.

FIG. 3 shows electrophoresis images of synthesized human lysozyme (an arrow indicates synthesized human lysozyme).

FIG. 4 shows specific activities of synthesized human lysozymes.

FIG. 5 shows the amounts of synthesized mIL6 protein.

FIG. 6 shows the amounts of synthesized DHFR protein.

FIG. 7 shows specific activities of synthesized human lysozymes (influence of PDI concentration).

FIG. 8 shows specific activities of synthesized human lysozymes (influence of DTT concentration in the presence of 0.13 μM PDI).

FIG. 9 shows activities of synthesized alkaline phosphatase (influence of GSSG concentration in the presence of 0.13 μM PDI and 1 mM DTT).

FIG. 10 shows activities of synthesized alkaline phosphatase (influence of DTT concentration difference in the presence of PDI).

FIG. 11 shows activities of synthesized lysozyme (influence of addition or no addition of DsbC).

FIG. 12 shows activities of synthesized alkaline phosphatase (comparison between the method of the present invention and a method using a cell extract).

FIG. 13 shows an electrophoresis image of EF-Tu elution fraction.

FIG. 14 shows an electrophoresis image of purified DsbA (lane 1) and DsbC (lane 2).

BEST MODE FOR CARRYING OUT THE INVENTION

The preferred embodiments of present invention will be hereafter described in greater detail, although the technical scope of the present invention is not limited thereto. Further, all modifications and variations are within the scope of the present invention.

The in vitro DNA transcription/translation system or RNA translation system of the present invention comprises a protein synthesis reagent comprising base components for protein synthesis caused by adding a template encoding a protein of interest such as mRNA, cDNA, or the like and (i) a reagent for conditioning a redox state and/or (ii) an enzyme catalyzing oxidation-reduction so as to artificially condition a state of redox equilibrium between disulfide and thiol.

[Method of Synthesizing a Protein Having Intermolecular or Intramolecular Disulfide Bonds]

The method of the present invention include a method of synthesizing a protein having intermolecular or intramolecular disulfide bonds, comprising using (1) a reaction system comprising a) and b) below, (2) a reaction system comprising a), b), and c) below, (3) a reaction system comprising a), b), and d) below, or (4) a reaction system comprising a), b), c), and d) below:

a) at least one template nucleic acid encoding a protein of interest;

b) a protein synthesis reaction reagent comprising a plurality of components that have been specified in terms of quantity and purity and causing a reaction to synthesize a protein encoded by a template nucleic acid upon addition of the template nucleic acid; c) at least one oxidoreductase catalyzing oxidation-reduction, which has been specified in terms of quantity and purity; and d) at least one redox reagent for conditioning a redox state, which has been specified in terms of quantity and purity. [1-1 Protein Synthesis Reagent]

The protein synthesis reagent is characterized in that it comprises, as components, predetermined amounts of ribosomes, initiation factors, elongation factors, termination factors, aminoacyl-tRNA synthetases, methionyl tRNA transformylases, tRNAs, amino acids, ribonucleoside triphosphates, 10-formyl 5,6,7,8-tetrahydrofolic acid (FD), salts, and water, which have been specified in terms of purity. However, it does not need to contain all components described above, so that it may selectively contain the components according to need.

The components used that constitute the system must not include a cell extract or crude fractions of such a cell extract. In addition, it is desired that the concentration of a substance influencing disulfide bond formation in proteins can be calculated.

In the reconstituted protein synthesizing system of the present invention, all components that constitute the system are reconstituted. Thus, it is easy to specify such components and calculate contents thereof.

The protein synthesis reaction reagent of the present invention can be used as a reaction system for protein synthesis in which DNA transcription/translation or RNA translation is carried out. In the present invention, the term “protein” indicates two or more amino acids binding to each other via peptide bonds, including peptides, oligopeptides, and polypeptides. The term “RNA” includes chemically synthesized RNA and mRNA. The term “DNA” indicates synthesized DNA, DNA vectors, genomic DNA, PCR products, and cDNA.

The condition that the protein synthesis reaction reagent of the present invention comprises components that have been specified in terms of quantity and purity refers to each component being independently purified such that the purity thereof can be calculated and the component can be quantified. In the present invention, the plurality of components that have been specified in terms of quantity and purity are substances that have been separately purified in advance via methods for purifying substances such as salting out, chromatography, electrophoresis, solubility difference, recrystallization, and centrifugation. The purities of such substances obtained via analysis methods using chromatography, electrophoresis, mass spectrometric analysis, centrifugation, and the like are about 80% or more and more preferably 90% or more. For instance, in the case of a protein, it is purified mainly via chromatography and the purity is measured via SDS-polyacrylamide gel electrophoresis (SDS-PAGE). In the case of a ribosome, it is purified mainly via ultracentrifugation and the purity is measured by sedimentation analysis via ultracentrifugation. A ribosome is an aggregated molecule having a molecular weight of several millions, comprising a plurality of RNA molecules (3 types of RNA molecules in prokaryotes: 23S, 5S, and 16S; and 4 types of RNA molecules in eukaryotes: 28S, 5.8S, 5S, and 18S) and a plurality of ribosomal proteins (about 50 proteins in prokaryotes and about 80 proteins in eukaryotes). As an aggregated molecule, it is subjected to sedimentation analysis so as to be identified and subjected to the measurement of the purity thereof. In addition, tRNAs are molecules that usually comprise 74 to 94 nucleotides and have a plurality of nucleotide sequences. Their purities can be measured by isolating the molecules via electrophoresis for identification and measuring absorbances at 260 nm and 280 nm. In addition, low-molecular weight substances such as amino acids and salts are identified via conventional methods such as chromatography, melting point measurement, elementary analysis, and mass spectrometric analysis such that the purities thereof can be measured.

Examples of factors and enzymes for transcription/translation as protein synthesis reagents that can be used include not only those derived from prokaryotic cells such as E. coli and but also those derived from eukaryotic cells:

(1) in the case of translation based on RNA, ribosomes, initiation factors, elongation factors, termination factors, aminoacyl-tRNA synthetases, tRNAs, adenosine triphosphate (ATP), guano sine triphosphate (GTP), amino acids, 10-formyl 5,6,7,8-tetrahydrofolic acid (FD), salts, and water are included, and methionyl tRNA transformylases are further included in the case of a reaction system derived from prokaryotic cells such as E. coli; and

(2) in the case of transcription/translation based on DNA, uridine triphosphate (UTP), cytidine triphosphate (CTP), and RNA polymerases such as T7RNA polymerase are included in addition to (1).

Various types of factors and enzymes constituting the reaction system of the present invention are originally contained in any organisms such as E. coli, molds, yeasts, and cultured cells. Thus, they can be separately purified at a high level so as to be used as components. However, recombinant products are preferably used because this allows each type of protein to be obtained in large volume, and unknown unnecessary or inhibitory components are less likely to be brought into the reaction system.

Specifically, genes encoding initiation factors, elongation factors, termination factors, aminoacyl-tRNA synthetases, methionyl tRNA transformylases, or RNA polymerases are ligated to adequate vectors, followed by transformation using E. coli, Bacillus subtilis, mold, yeast, or the like for expression induction. Then, the proteins are purified such that components constituting the reaction system of the present invention can be obtained. When various types of factors and enzymes are produced using transformants, proteins may be expressed as being intact or may be expressed as fusion proteins. Examples of a fusion protein include a Histidine-Tag (hereafter referred to as His-Tag), a strept-Tag, a GST-Tag, and a FLAG-Tag (Appl Microbiol Biotechnol. 60 (5):523-533 (2003)).

Various forms of methods for purifying His-Tagged protein components using a His-Tag and a nickel column have been known can be selectively used according to need. As an example, an outline of such a method is described as follows:

1. obtaining a fusion protein by allowing a His-Tag (comprising 6 His) to bind at the N-terminal of a protein of interest via a genetic engineering technique;

2. sonicating cells in which His-Tagged proteins have been expressed in ice and allowing the cells be suspended in a loading buffer (300 mM NaCl, 50 mM NaH₂PO₄, pH 8.0);

3. allowing the cell lysate to be subjected to centrifugation (30,000 g, 4° C., 30 minutes);

4. adding 50% Ni²⁺-NTA slurry (Qiagen) equilibrated with an ice-cooled loading buffer to the supernatant obtained above, followed by stirring at 4° C. for 1 hour;

5. loading resin into a column, followed by washing using a loading buffer in a volume 20 times that of the column at 4° C.;

6. washing the column using a loading buffer (containing 10 mM imidazole, pH 8.0) in a volume 20 times that of the column at 4° C.; and

7. setting the imidazole concentration gradient to 10 to 250 mM using a loading buffer in a volume 20 times that of the column, allowing the protein of interest to be eluted from the column, collecting fractions (1 ml each), and confirming the protein of interest via SDS-PAGE.

More preferably, after purification of the protein as described above, the following enzymes that are not directly involved in protein synthesis are added to the reaction system of the present invention as various types of factors and enzymes constituting the reaction system: enzymes related to energy regeneration such as creatine kinases, myokinases, and nucleoside diphosphate kinases; and enzymes used for degradation of inorganic pyrophosphoric acids generated via a transcription/translation reaction, such as inorganic pyrophosphatases.

Herein, it is essential for salts to contain cations and anions that are necessary for transcription/translation. Examples thereof that are generally used include potassium glutamate, ammonium chloride, magnesium acetate, and calcium chloride. Needless to say, in addition to the salts described above, appropriate salts can be selectively used. Examples of water include types that do not contain ions, microorganisms, enzymes, and the like, such as water produced using a Milli-Q system (Millipore) and commercially available pure water.

Ribosomes are sites for peptide synthesis. When a ribosome binds to mRNA, aminoacyl-tRNA is located at an A site and formyl methionyl tRNA or peptidyl tRNA is located at a P site in the ribosome, such that a reaction of forming peptide bonds takes place (Science 289:920-930 (2000)). In the present invention, ribosomes having such functions can be used regardless of their origins. Examples thereof that can be used include E. coli-derived ribosomes and eukaryotic cell-derived ribosomes. Preferably, ribosomes that can be used in the present invention are those derived from E. coli such as E. coli A19 cells and E. coli MRE600 cells.

As initiation factors of the in vitro protein synthesizing system of the present invention, those derived from E. coli such as IF1, IF2, and IF3 have been known, which are essential for formation of a translation initiation complex or act as factors significantly promoting such formation (Biochemistry 29:5881-5889 (1990)). The initiation factor IF3 promotes dissociation of a 70S ribosome into a 30S subunit and a 50S subunit, which is a necessary step for translation initiation. In addition, it inhibits insertion of tRNA excluding formyl methionyl tRNA at the P site upon translation initiation complex formation. The initiation factor IF2 binds to formyl methionyl tRNA and transfers formyl methionyl tRNA to the P site of a 30S ribosome subunit, resulting in formation of a translation initiation complex. The initiation factor IF1 promotes functions of initiation factors IF2 and IF3. Preferred examples of initiation factors that can be used in the present invention include those derived from E. coli such as E. coli K12 cells. In addition, those derived from eukaryotic cells can also be used.

There are two types of EF-Tu elongation factors: GTP and GDP. GTP binds to aminoacyl-tRNA so as to transport the tRNA to the A site of a ribosome. When EF-Tu is leaving the ribosome, GTP is hydrolyzed so as to be converted into GDP (EMBO J. 17: 7490-7497 (1998)). Meanwhile, an EF-Ts elongation factor binds to EF-Tu (GDP) so as to promote conversion of GDP into GTP (Archives of Biochemistry and Biophysics 348: 157-162 (1997)). In addition, an EF-G elongation factor promotes a translocation reaction after a reaction of peptide bond formation during the process of peptide chain elongation (Nature Structure Biology 6:643-647 (1999); FEMS Microbiology Reviews 23:317-333 (1999)). Preferred examples of elongation factors used in the present invention are those derived from E. coli, such as those obtained from E. coli K12 cells. In addition, those derived from eukaryotic cells can also be used.

Termination factors are essential for ribosome recycling during the process of termination of protein synthesis, dissociation of peptide chains subjected to translation, and initiation of subsequent mRNA translation. When protein synthesis is carried out in a reaction system without termination factors, the reaction terminates before a termination codon so that a stable complex of a ribosome, a peptide, and mRNA is easily formed (a polysome display method, a ribosome display method, and an in vitro virus method). In addition, introduction of an unnatural amino acid into a peptide chain is carried out by omitting RF1 and/or RF2 from the reaction system. In other words, introduction of unnatural amino acid into a UAG codon and a UGA codon is carried out with high efficiency when RF1 and RF2 are omitted, respectively.

When a termination codon (UAA, UAG, or UGA) is located at the A site of a ribosome, termination factors RF1 and RF2 move to the A site so as to promote dissociation of a peptide chain from peptidyl tRNA (located at the P site). RF1 recognizes termination codons UAA and UAG, and RF2 recognizes termination codons UAA and UGA. A termination factor RF3 promotes dissociation of RF1 and RF2 from a ribosome after a reaction of peptide chain dissociation caused by RF1 and RF2. A ribosome recycling factor (RRF) promotes detachment of tRNA remaining at the P site after protein synthesis termination and ribosome recycling for subsequent protein synthesis. In the present invention, RRF is regarded as a termination factor. In addition, functions of RF1, RF2, RF3, and RRF termination factors are explained in EMBO J. 16: 4126-4133 (1997) and EMBO J. 16: 4134-4141 (1997). Preferred examples of termination factors that can be used in the present invention are those derived from E. coli such as those obtained from E. coli K12 cells. In addition, those derived from eukaryotic cells can also be used.

An aminoacyl-tRNA synthetase is an enzyme synthesizing aminoacyl-tRNA by causing covalent binding of an amino acid and tRNA in the presence of ATP (RNA 3:954-960 (1997); Protein, Nucleic Acid and Enzyme, 39: 1215-1225 (1994)). Preferred examples of an aminoacyl-tRNA synthetase that can be used in the present invention are those derived from E. coli, such as those obtained from E. coli K12 cells. In addition, those derived from eukaryotic cells can also be used. Further, an artificial aminoacyl-tRNA synthetase recognizing an unnatural amino acid can also be used (JP Patent No. 2668701).

A methionyl tRNA transformylase (MTF) is an enzyme synthesizing N-formyl methionyl (fMet) tRNA in which a formyl group has bound to an amino group of methionyl tRNA as a result of protein synthesis in prokaryotes. That is, methionyl tRNA transformylase causes a formyl group of N10-formyl tetrahydrofolic acid to be transferred to the N-terminal of methionyl tRNA corresponding to an initiation codon such that fMet-tRNA is formed (Proc. Natl. Acad. Sci. USA, 96: 875-880 (1999)). The thus added formyl group is recognized by an initiation factor IF2 so that it can function as an initiation signal of protein synthesis. MTF does not exist in a synthesis system of eukaryotic cytoplasm; however, it exists in synthesis systems of eukaryotic mitochondria and chloroplast. Preferred examples of MTF that can be used in the present invention are those derived from E. coli, such as those obtained from E. coli K12 cells.

RNA polymerase is an enzyme transcribing a DNA sequence to RNA, and it has been known to exist in various types of organisms. An example thereof is T7 RNA polymerase that is derived from T7 phage. This polymerase is an enzyme that binds to a specific DNA sequence called a T7 promoter so as to transcribe the downstream DNA sequence to RNA. The inventors of the present invention added a His-Tag to the N-terminal of T7 RNA polymerase, caused massive expression of the polymerase as a fusion protein in an E. coli BL21 cell line, and carried out purification via affinity chromatography using a nickel column. In the present invention, as well as T7 RNA polymerase, various types of RNA polymerases can be used. For instance, T3 RNA polymerase and SP6 RNA polymerase have been commercially available, and they can be used in the present invention.

Examples of amino acids include natural or unnatural amino acids and tRNAs charged with natural or unnatural amino acids. When using such a tRNA charged with an unnatural amino acid, an unnatural amino acid can be introduced into a protein.

Examples of tRNA that can be used include tRNA purified from cells of E. coli, yeast, and the like. In addition, artificial tRNA in which an anticodon or another base is optionally modified (J. Am. Chem. Soc. 121:34-40 (1996), Nature Biotech. 20: 177-182 (2002)) can be used. For instance, when tRNA having CUA as an anticodon is charged with an unnatural amino acid, a UAG codon that is a termination codon in its original form can be translated to an unnatural amino acid. Using this method, an unnatural amino acid can be introduced into a protein in a site-specific manner.

Further, as a buffer, a potassium phosphate buffer (pH 7.3) is generally used.

[1-2 Substances Influencing Disulfide Bond Formation]

Examples of substances influencing disulfide bond formation include an oxidoreductase that is an enzyme catalyzing oxidation-reduction of a disulfide bond and/or a redox reagent that is a reagent for conditioning a redox state of a disulfide bond. More specifically, examples of an enzyme and/or reagent influencing disulfide bond formation include: (i) enzymes catalyzing oxidation-reduction such as proteins, including glutathion reductases, thioredoxin reductases, protein disulfide isomerases, disulfide interchange proteins, and thioredoxin-like proteins; and/or (ii) reagents for conditioning a redox state such as low-molecular-weight compounds, including reduced glutathione, oxidatized glutathione, DTT, 2-mercaptoethanol, and thioredoxin. The term “redox reagent” of the present invention indicates a reagent that can reduce disulfide to thiol or can oxidate thiol so as to form disulfide.

Preferably, an enzyme and/or a substrate thereof promoting/regulating disulfide bond crosslinking in proteins is used as (i) an oxidoreductase (enzyme catalyzing oxidation-reduction) and/or (ii) a redox reagent (reagent for conditioning a redox state), which are substances influencing disulfide bond formation. In addition, (i) an enzyme catalyzing oxidation-reduction and/or (ii) a reagent for conditioning a redox state are not necessarily added during a translation reaction. They may be added after the termination of translation. When they are added after the termination of translation, preferably, they are allowed to stand for several tens of minutes to about an hour at 37° C. after addition.

When DTT is used as a reagent for conditioning a redox state, the concentration thereof is 0 to 1 mM, preferably 0.001 to 0.5 mM, and more preferably 0.060 to 0.5 mM.

When oxidized glutathione is used as a reagent for conditioning a redox state, the concentration thereof is 0 to 8 mM, preferably 0.1 to 4 mM, and more preferably 1 to 4 mM.

In accordance with the method for protein synthesis described above, disulfide interchange proteins are preferably DsbA and/or DsbC.

Protein disulfide isomerase (EC 5.3.4.1.) is an enzyme having a size of about 55 kDA, existing in endosporium of the endoplasmic reticulum of a eukaryote, and catalyzing formation, isomerization, and/or reduction reaction of a disulfide bond. It is considered to have chaperone-like activity. As mentioned above, this protein can be used as a component after being purified from organisms. Also, it may be obtained through recombination production for use. For instance, protein disulfide isomerase (PIR database accession no. ISBOSS) (SEQ ID NO: 5) purified from the liver of a bovine or purified proteins obtained through recombinant expression of a protein disulfide isomerase gene of yeast using E. coli can be used.

In addition, the following proteins have been known as protein disulfide isomerases and they can be used as protein disulfide isomerases of the present invention:

human protein disulfide-isomerase (PIR database accession no. ISHUSS) (SEQ ID NO: 6);

human protein disulfide-isomerase-related protein (GenBank accession no. 4758304) (SEQ ID NO: 7); and

yeast protein disulfide isomerase homolog (PIR database accession no. A44483) (SEQ ID NO: 8).

As disulfide interchange proteins, four types of proteins called disulfide interchange proteins A, B, C, and D (DsbA, B, C, and D) have been known to exist in E. coli and the like. DsbA is an enzyme having a 21-kDa thioredoxin-like fold structure, and it is considered to catalyze disulfide bond formation. DsbB is a protein 20 kDa in size, which has four transmembrane regions and two periplasm domains. DsbB is considered to maintain DsbA in a state of being oxidized. DsbC is a periplasm protein that forms a homodimer and has a thioredoxin-like fold. DsbC is considered to be an enzyme that is mainly responsible for disulfide bond isomerization and to function as chaperone. DsbD is a protein having a molecular weight of 59 kDa, which comprises two periplasm domains and eight transmembrane regions. DsbD is considered to have a function of maintaining cysteine as an active center of DsbC in a state of being reduced.

The following proteins have been known as disulfide interchange proteins, and they can be used as disulfide interchange proteins of the present invention:

E. coli DsbA (SWISS-PROT protein database accession no. P24991) (SEQ ID NO: 9);

E. coli DsbC (SWISS-PROT protein database accession no. P21892) (SEQ ID NO: 10);

Salmonella typhimurium protein disulfide-isomerase dsbA homolog (PIR database accession no. S32895) (SEQ ID NO: 11);

Neisseria meningitidis thiol-disulfide interchange protein dsbA homolog NMB0278 (PIR database accession no. C81217) (SEQ ID NO: 12);

Caenorhabditis elegans protein disulphide isomerase isoform I (GeneBank accession no. AAB94647) (SEQ ID NO: 13); and

Datisca glomerata protein disulfide isomerase homolog (GeneBank accession no. AAD28260) (SEQ ID NO: 14).

As mentioned before, these disulfide interchange proteins can be purified from organisms so as to be used as components, and also, they may be obtained through recombination production for use.

When using protein disulfide isomerase (PDI) as oxidoreductase, the concentration thereof is preferably 0 to 10 μM, more preferably 0.001 to 5 μM, and further preferably 0.001 to 2 μM. When using disulfide interchange protein as oxidoreductase, the concentration thereof is preferably 0 to 10 μM, more preferably 0.01 to 10 μM, and further preferably 0.1 to 10 μM.

In addition, preferably, disulfide interchange proteins contain minimum amounts of glutathion reductases and thioredoxin reductases. Preferably, the contents of thioredoxin reductase and/or glutathion reductase in a component constituting a protein synthesizing system are 100 ng/ml or less.

In addition, a method of measuring the purity of each component described above is exemplified below.

The purity can be calculated in the following manner: the aforementioned components comprising proteins such as initiation factors, elongation factors, termination factors, methionyl tRNA transformylases, DsbA, and DsbC are purified by a method of purifying His-Tagged proteins using a His-Tag and a nickel column; proteins of interest are confirmed by SDS-PAGE; and the electrophoresis pattern of each lane is using a densitometer.

The purity of purified ribosomes can be measured via sucrose density-gradient analysis.

Reagents that have been commercially available in general such as tRNAs, amino acids, ribonucleotide triphosphates, FD, other types of buffers, DTT, and oxidized glutathione can be used at the purity level of commercially available forms of such reagents. In addition, all such commercially available reagents have a purity of 80% or more.

[Test Method and Synthesis Method Utilizing the Same]

As a test method of the present invention, the reaction system of the [method of synthesizing a protein having intermolecular or intramolecular disulfide bonds] described above can be used.

The test method of the present invention comprises measuring correlations among the concentration of an oxidoreductase, the concentration of a redox reagent, and the activity of a synthesized protein comprising at least one peptide chain, in which crosslink formation can be carried out using a single disulfide bond, and the activity of which is controlled based on the pattern of such crosslink formation. The method is carried out using a reaction system comprising the following a) to d), and specifically (1) a), b), and c), (2) a), b), and d), and (3) a), b), and c), and d):

a) at least one template nucleic acid;

b) a protein synthesis reaction reagent comprising a plurality of components that have been specified in terms of quantity and purity and causing a reaction to synthesize a protein encoded by a template nucleic acid upon addition of the template nucleic acid; c) at least one oxidoreductase catalyzing oxidation-reduction, which has been specified in terms of quantity and purity; and d) at least one redox reagent for conditioning a redox state, which has been specified in terms of quantity and purity.

The activity of a protein synthesized via the above reaction system is measured such that correlation among the protein, c) the oxidoreductase added, and d) the redox reagent added is determined. Herein, the protein activity to be measured is not limited to enzyme activity. For instance, binding activity between a receptor and a protein, cellular proliferation activity, specific activity between binding activity and cellular proliferation activity, and the like are included.

More specifically, c) oxidoreductases and d) redox reagents at various concentrations are prepared, upon which c) and d) may each comprise a single substance or may be a mixture of a plurality of substances. Examples of such oxidoreductases include protein disulfide isomerases, disulfide interchange proteins, and homologous enzymes. Examples of such redox reagents include DTT, GSSG, GSH, and thioredoxin. Subsequently, a protein synthesis reaction is initiated. Subsequently, a protein synthesis reaction is carried out. Herein, c) and d) may be added at the beginning of the protein synthesis reaction, may be added during the synthesis reaction, or may be added after the termination of the synthesis reaction. When c) and d) are added after the termination of the synthesis reaction, the resultant is preferably allowed to stand for several tens of minutes to an hour at about 37° C. after the addition. In each case of protein synthesis described above, the activity of a protein synthesized in a reaction solution is determined. Herein, a protein to be synthesized is not particularly limited, as long as it is a protein in which crosslink formation of at least one intramolecular and/or intermolecular disulfide bond can be achieved. For instance, a plurality of proteins are allowed to be synthesized in a single reaction solution so as to form a heterooligomer or the like, such that the activities thereof can be determined.

As described above, correlation of protein activity, c) an oxidoreductase, and/or d) a redox reagent can be revealed.

With the use of the thus obtained correlation, a protein of interest can be created with efficiency in a reaction system produced by: determining concentrations of c) an oxidoreductase and/or d) a redox reagent that are necessary to obtain a predetermined activity level (desired activity level); adding c) an oxidoreductase and/or d) a redox reagent to b) a protein synthesis reaction reagent so as to achieve a concentration at which the desired activity can be obtained; and adding a) a template nucleic acid encoding the protein of interest.

The present invention is hereafter described in greater detail with reference to the following examples, although the technical scope of the present invention is not limited thereto.

Example 1 Ribosome Preparation

Cells of an E. coli A19 cell line (300 g) during the middle logarithmic phase were disrupted using alumina particles. The thus disrupted cells were suspended in a buffer A (10 mM HEPES-potassium hydroxide (pH 7.6, HEPES-KOH), 10 m Magnesium chloride (MgCl₂), 50 mM potassium chloride (KCl), and 1 mM DTT). Then, alumina particles and disrupted cells were removed therefrom via centrifugation (30,000 g, 4° C., 1 hour). Then, deoxyribonuclease (DNase) was added to the thus obtained supernatant fraction to a final concentration of 1 μg/ml, followed by centrifugation (100,000 g, 4° C., 4 hours). The resulting pellet was suspended in a buffer A such that a crude ribosome extract was prepared. The crude ribosome extract was subjected to 6% to 36% (w/v) sucrose density-gradient centrifugation. A fraction shown in FIG. 1 resulted from fractionation. The obtained ribosome fraction was subjected to centrifugation at 100,000 g. The resulting pellet was suspended in a ribosome buffer (20 mM HEPES-KOH (pH 7.6), 6 mM MgOAc, 30 mM NH₄Cl, 7 mM β-mercaptoethanol (mercaptoethanol)) such that purified ribosomes were prepared. Some of purified ribosomes were subjected to 6% to 36% (w/v) sucrose density-gradient analysis such that a single peak was formed. The purity of the ribosomes was 90% or more.

Example 2 Construction of Plasmids Hyperexpressing Aminoacyl-tRNA Synthetase (ARS) and Production of Transformants

Using a genome extracted from an E. coli A19 cell line as a template, a gene sequence encoding alanyl-tRNA synthetase was amplified by PCR such that a DNA fragment having a sequence recognized by SphI at the 5′ end and HindIII at the 3′ end was obtained. The obtained DNA fragment was inserted into a plasmid pQE30 (QIAGEN) that had previously been cleaved with SphI and HindIII. Thus, a vector used to cause high expression of alanyl-tRNA synthetase having a His-Tag fused at the N-terminal thereof was obtained. The thus obtained vector was used for transformation of E. coli BL21/pREP4. Another vector hyperexpressing ARS was constructed in a similar manner. Table 1 lists vectors, restriction enzymes, and sites of His-Tag. In addition, a series of pQE plasmids and a series of pET plasmids listed in table 1 were used for transformation of E. coli BL21/pREP4 and E. coli BL21/DE3, respectively.

TABLE 1 Enzymes or factors Vector N-terminal R. E. C-terminal R. E. Site of His-Tag AlaRS pQE30 SphI HindIII N ArgRS pET16b NdeI BamHI N AsnRS pQE30 BamHI HindIII N AspRS pET21a NdeI XhoI C CysRS pET21a NdeI XhoI C GlnRS pET21a NdeI XhoI C GluRS pET21a NdeI XhoI C GlyRS pET21a NdeI XhoI C HisRS pET21a NdeI XhoI C IleRS pET21a NdeI HindIII N LeuRs pET21a XbaI XhoI C LysRS pET21a NdeI XhoI C MetRS pET21a XbaI XhoI C PheRS pQE30 SphI HindIII N ProRS pET21a NdeI XhoI C SerRS pET21a XbaI XhoI C ThrRS pQE30 BamHI HindIII N TrpRS pET21a NdeI XhoI C TyrRS pET21a NdeI XhoI C ValRS pET21a XbaI NotI C MTF pET21a NdeI XhoI C IF1 pQE30 BamHI HindIII N IF2 pQE30 BamHI HindIII N IF3 pQE30 BamHI HindIII N EF-G pQE60 MunI BglII C EF-Tu pQE60 EcoRI BglII C EF-Ts pQE60 NcoI BamHI C RF1 pQE60 BamHI HindIII C Note: “R. E.” stands for restriction enzyme.

Example 3 Construction of Other Plasmids Hyperexpressing Protein Factors and Enzymes

The following plasmids hyperexpressing protein factors and enzymes were constructed in a manner similar to that described in Example 2: MTF, T7 RNA polymerase, IF1, IF2, IF3, EF-G, EF-Tu, EF-Ts, and RF1. In addition, plasmids for protein factors and enzymes that are not listed in table 1 were also constructed in a similar manner. In addition, a series of pQE vectors or a series of pET vectors were used for transformation of E. coli BL21/pREP 4 or E. coli BL21/DE3.

Example 4 Hyperexpression and Purification of Protein Factors and Enzymes

In order to cause hyperexpression of His-Tagged alanyl-tRNA synthetase, E. coli BL21/pREP4 cells that were transformants obtained in Example 2 were cultured in an LB medium (6 liter) until the absorbance at 660 nm became 0.7. To the culture solution, isopropyl-1-thio-β-D-galactopyronoside (IPTG) was added to a final concentration of 0.1 mM, followed by additional culture at 37° C. for 4 hours. The culture solution was subjected to centrifugation and the obtained cells were suspended in a suspension buffer (50 mM HEPES-KOH (pH 7.6), 1 M NH₄Cl, 10 mM MgCl₂, 0.3 mg/ml albumen lysozyme, 0.1% TritonX-100, 0.2 mM phenylmethanesulfonyl fluoride (PMSF), and 6 mMβ-mercaptoethanol). The suspension was sonicated such that the cells were disrupted. The sonicated suspension was subjected to centrifugation (100,000 g, 4° C., for 1 hour). Then, disrupted cells were removed therefrom. The obtained supernatant fraction was placed in a 10-ml Hi-Trap chelating column (Pharmacia) that had been precharged with Ni²⁺, followed by washing with 100 ml of an HT buffer containing 10 mM imidazole (50 mM HEPES-KOH (pH 7.6), 1M NH₄Cl, and 10 mM MgCl₂). His-Tagged alanyl-tRNA synthetase was eluted from the column with a linear gradient of 10 to 400 mM imidazole concentration in the HT buffer. Fractions containing purified proteins were dialyzed using a stock buffer (50 mM HEPES-KOH (pH 7.6), 100 mM KCl, 10 mM MgCl₂, and 30% glycerol). The concentration of purified His-Tagged alanyl-tRNA synthetase was calculated based on a calibration curve prepared using a Protein Assay Kit (Bio-Rad) with reference to bovine serum albumin (BSA). The purified His-Tagged alanyl-tRNA synthetase was aliquoted at 1 ml and rapidly frozen using liquid nitrogen, followed by storage at −80° C. His-Tagged alanyl-tRNA synthetase (AlaRS), arginine tRNA synthetase (ArgRS), asparagine tRNA synthetase (AsnRS), aspartic acid tRNA synthetase (AspRS), cysteine tRNA synthetase (CysRS), glutamine tRNA synthetase (GlnRS), and glutamic acid tRNA synthetase (GluRS) were purified in a similar manner. FIG. 2 shows separation of His-Tagged factors via 12% SDS-PAGE (subjected to Coomassie brilliant blue staining). The purity of each factor calculated by a densitometer was 90% or more. In addition, other factors and enzymes of table 1, which are not shown in FIG. 2, were also subjected to separation by SDS-PAGE. The purity of each factor or enzyme calculated by a densitometer in a similar manner was 90% or more.

Example 5 Translation Experimentation General Methods

The composition of a protein synthesis reaction reagent (50 μl) was as follows: 2 mM of ATP, 2 mM of GTP, 1 mM of CTP, 1 mM of UTP, 10 mM of creatine phosphate (creatine phosphate), 2.8 A 260 unit tRNA mix, 0.5 μg of FD, 0.1 mM of each amino acid, 9 mM of magnesium acetate, 5 mM of potassium phosphate (pH 7.3), 95 mM of potassium glutamate, 5 mM of ammonium chloride, 0.5 mM of calcium chloride, 1 mM of spermidine, 8 mM of putrescine, 12 pmol of ribosome, 1 μg of IF1, 2 μg of IF2, 0.75 μg of IF3, 1 μg of EF-G, 2 μg of EF-Tu, 1 μg of EF-Ts, 0.5 μg of RF1, 0.5 μg of RF3, 0.5 μg of RRF, 30-300 units each of ARS and MTF, 0.2 μg of creatine kinase (CK), 0.15 μg of myokinase (MK), 0.054 μg of nucleoside diphosphate kinase (NDK), 1.78 units of PPiase, and 0.5 μg of T7 RNA polymerase. To this protein synthesis reaction reagent, 1 pmol of template DNA was added, followed by reaction at 37° C. for 1 hour.

When it was necessary to remove ribosomes after reaction, ribosomes were removed by allowing them to pass through an ultrafilter membrane through which substances having sizes of 100 kDa or less can pass.

In addition, ribosomes and items listed in table 1 among the items of the above composition were prepared in the manner described in Examples 1 and 4, and the purities thereof were measured. The other components used were commercially available purified reagents.

Also, when the composition described above was applied for Examples 6 to 12 described below, items listed in table 1, ribosomes, DsbA, and DsbC were prepared in the manner described in Examples 1, 4, and 13, and the purities thereof were measured. The other components used were commercially available purified reagents.

Example 6 Synthesis of Human Lysozyme

It has been reported that lysozyme is a protein that originally has four intramolecular disulfide bonds and that two or more of such disulfide bonds must be crosslinked with each other such that the activity thereof is exhibited (J Biol Chem 251:3147-3153 (1976)). Thus, lysozyme has long been widely used as a model protein with which correlation between the structure and the activity of a protein is examined. Herein, in accordance with the method of the present invention and a conventional method, human lysozyme was synthesized such that the amounts and specific activities of proteins synthesized could be examined.

From a human lysozyme cDNA clone (human gene collection, Stratagene), the following two primers were used such that a gene corresponding to matured human lysozyme having a size of 0.42 kbp was amplified by PCR: the forward primer sequence: AAGGAGATATACCAATGAAGGTCTTTGAAAGGTGTG (SEQ ID NO: 1); and the reverse primer sequence: GGATTAGTTATTCATTACACTCCACAACCTTGAACAT (SEQ ID NO: 2).

Then, with the use of a forward primer having a sequence of GAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTT TGTTTAACTTTAAGAAGGAGATATACCA (SEQ ID NO: 3) and the above reverse primer, template DNA about 0.51 kbp in length containing a T7 promoter region was amplified by PCR. The sequence of the template DNA is set forth in SEQ ID NO: 4 in the Sequence Listing.

To

protein synthesis reaction reagent (used in the method of the present invention) described in Example 5 and 50 μl of a reaction liquid obtained by adding 1 mM of DTT to the reaction reagent (used in the conventional method), 1 pmol of prepared template DNA was added such that a protein synthesis reaction was carried out.

Synthesized proteins were separated by SDS-PAGE so as to be subjected to staining with SYPRO Orange (Amersham pharmacia). A band of human lysozyme having a molecular weight of 17000 was compared with the band of BSA having a known concentration such that the concentration of synthesized human lysozyme was obtained. The activity of human lysozyme was measured using frozen dry powders of a Micrococcus luteus ATCC 4698 cell line (SIGMA) under the condition that the amount that can cause a decrease in absorbance at 450 nm by 0.001 per minute was determined to be 1 unit (Imoto T., Johnson L. N., North A. T. C., Phillips D. C., Rupley J. A., The Enzymes, 3rd ed., 7, 665-868 (1972)). FIGS. 3 and 4 show an electrophoresis image and specific activities of synthesized proteins, respectively. As is apparent from FIGS. 3 and 4, in the case of the conventional method, synthesized proteins were confirmed via electrophoresis; however, no activity was confirmed. On the other hand, in the case of the method of the present invention, specific activity at about 300 units/mg was confirmed. Thus, it was found that, in accordance with the present invention, folding of a protein originally having disulfide bonds was properly carried out. Also, other proteins having disulfide bonds were examined so that substantially equivalent results were obtained.

Example 7 Synthesis of Murine Interleukin 6 (mIL 6)

With the use of a mouse cDNA library, mIL6 was prepared in a manner similar to that used for Example 6. In addition, template DNA of E. coli. dihydrofolate reductase (DHFR) was prepared using E. coli. genomic DNA in a manner similar to that used for Example 6. Then, the following five types of reaction solutions were prepared: a reaction solution obtained by adding 1 mM of DTT to the protein synthesis reaction reagent of Example 5 (the conventional method); the protein synthesis reaction reagent of Example 5; and reaction solutions obtained by separately adding GSSG at final concentrations of 2, 4, and 8 mM to the reagent of Example 5. To each reaction solution, 1 pmol of template DNA was added, such that proteins corresponding to the respective reaction solutions were synthesized. In the case of DHFR synthesis, the reaction solutions were allowed to pass through ultrafilter membranes after protein synthesis. Then, the reaction solutions of DHFR synthesis and the reaction solutions of mIL6 synthesis were separately subjected to SDS-PAGE, followed by fluorescent staining of proteins with SYPRO Red. Staining patterns were analyzed using a fluoroimager so that amounts of proteins in the reaction solutions could be calculated based on color depth of mIL6 and that of DHFR. FIGS. 5 and 6 show the amounts of mIL6 proteins obtained and those of DHFR proteins obtained, respectively. As is apparent from FIGS. 5 and 6, when DHFRs having no intramolecular disulfide bonds were synthesized, the largest amounts of proteins were produced by the conventional method and the amounts of protein synthesized by the method of the present invention were less. On the other hand, it was confirmed that, when mIL6 having intramolecular disulfide bonds was synthesized, the amounts of proteins produced by the method of the present invention exceeded those produced by the conventional method. In addition, the same experiment was carried out except that template DNA of a different protein having disulfide bonds was used instead of template DNA of mIL6. Accordingly, substantially equivalent results were obtained.

Example 8 Influence of Protein Disulfide Isomerase (PDI)

Four reaction solutions were prepared, each of which contained the protein synthesis reaction reagent of Example 5 with addition of 2 mM GSSG. Then, bovine-derived PDI (Takara Bio Inc.) was added to the reaction solutions to final concentrations of 0, 0.0325, 0.13, and 0.52 μM, respectively. Thereafter, template DNA of human lysozyme (1 pmol) was added to 50 μl of each reaction solution, resulting in protein synthesis. The specific activities of lysozymes were determined in a manner similar to that used for Example 6. FIG. 7 shows relationships between concentrations of PDI added to the reaction systems and specific activities of human lysozymes synthesized. As is apparent from FIG. 7, specific activities of proteins having disulfide bonds were found to be improved with addition of PDI to a concentration of 0.0325 μM or more. Further, it was confirmed that PDI activity can be determined by the method of the present invention. In addition, when other types of PDIs and disulfide interchange proteins were used instead of bovine PDI, substantially equivalent results were obtained.

Example 9 Influence of Dithiothreitol (DTT) Concentration

Six reaction solutions were prepared, each of which contained the protein synthesis reaction reagent of Example 5 with addition of 0.13 μM PDI. Then, DTT was added to the reaction solutions to final concentrations of 1000, 500, 250, 125, 62.5, and 0 μM, respectively. Thereafter, 1 pmol of template DNA of human lysozyme was added to 50 μl of each reaction solution, resulting in protein synthesis. The specific activities of lysozymes were determined in a manner similar to that used for Example 6. FIG. 8 shows the results. As is apparent from FIG. 8, it was revealed that, with a DTT concentration of 125 μM or less, strong lysozyme activity can be obtained.

Example 10 Synthesis of Alkaline Phosphatase

Alkaline phosphatase is a protein having two intramolecular disulfide bonds, and thus it has long been deeply examined as a model protein with regard to the relationship between disulfide bond formation and the enzyme activity. Herein, alkaline phosphatase was synthesized.

PCR primers specific to E. coli genomic DNA and E. coli. alkaline phosphatase were used to produce template DNA of E. coli. alkaline phosphatase in a manner similar to that used for Example 6. Five reaction solutions were prepared, each of which contained the protein synthesis reaction reagent of Example 5 with the addition of 1 mM DTT and 0.13 μM PDI. Then, GSSG was added to the reaction solutions to concentrations of 0, 1, 2, 3, and 4 mM, respectively.

Thereafter, 1 pmol of template DNA of alkaline phosphatase gene was added to 50 μl of each reaction solution, resulting in protein synthesis. Concentrations of synthesized proteins were measured in a manner similar to that used for Example 5. Activities of synthesized alkaline phosphatases were determined by measuring the absorbance at 405 nm using p-nitrophenyl phosphate disodium salt as a substrate (Biochim Biophys Acta. 258:178-87 (1972). FIG. 9 shows relative activities of alkaline phosphatases. As is apparent from FIG. 9, no activity of alkaline phosphatase was obtained by the conventional method; that is to say, with the use of a protein reaction reagent with addition of 1 mM DTT and 0.13 μM PDI. On the other hand, the activity of alkaline phosphatase was obtained by the method of the present invention; that is to say, with the use of a reaction solution with addition of 1 mM or 2 mM GSSG. Even with the presence of 1 mM DTT in a system, the activity of alkaline phosphatase was confirmed. Thus, the occurrence of proper folding of synthesized proteins was revealed. In addition, different reaction solutions were prepared, containing the reaction solution of Example 5 and DTT at concentrations of 1000, 500, 250, 125, and 0 μM, respectively. As described above, template DNA was added to each reaction solution, followed by protein synthesis. Then, activities of alkaline phosphatase were determined. The results are shown in FIG. 10. In the absence of PDI, DTT concentrations were exclusively changed: Accordingly, it was revealed that the activity of alkaline phosphatase was obtained at a DTT concentration of 500 μM or less.

Example 11 Influence of DsbC

E. coli DsbC was amplified by PCR and a vector hyperexpressing dsbC was constructed in a manner similar to that used for Example 2, except that a DNA fragment having a sequence recognized by BamHI at the 5′ end and HindIII at the 3′ end was obtained. With the use of the obtained vector, E. coli. BL21/pREP4 was transformed. Then, purified DsbC was obtained in a manner similar to that used for Example 4. The protein purity was 90% or more. Three reaction solutions (50 μl each) were prepared by adding 1 mM DTT and 2 mM GSSG to the protein synthesis reagent of Example 5. To these reaction solutions, DsbC and template DNA of the human lysozyme gene of Example 6 were added at concentrations of 0, 0, and 0.5 μM and at concentrations of 0, 1, and 1 pmol, respectively. Amounts of synthesized proteins and activities of lysozyme were determined in a manner similar to that used for Example 6. FIG. 11 shows the specific activities. As is apparent from FIG. 11, it was revealed that addition of DsbC causes significant increase in the specific activity of lysozyme. In addition, when other disulfide interchange proteins were used instead of DsbC, and also when different types of disulfide interchange proteins were simultaneously used, substantially equivalent results were obtained.

Example 12 Comparison Between the Protein Synthesis Reagents of the Present Invention and Conventional Cell Extracts

In accordance with the manufacturer's instructions, E. coli alkaline phosphatase was synthesized, except that GSSG was added to final concentrations of 0, 1, 2, and 3 mM using an in vitro protein synthesizing system (Roche Rapid Translation System 100) in which a cell extract of E. coli was used. In addition, E. coli alkaline phosphatase was synthesized by the method of the present invention in a manner similar to that used for Example 10. FIG. 12 shows specific activities of synthesized alkaline phosphatases. As is apparent from FIG. 12, in the system using a cell extract of E. coli, no activity of alkaline phosphatase was found, even with addition of GSSG at various concentrations, while on the other hand, the activity of alkaline phosphatase at a high level was confirmed by the synthesis method of the present invention.

Example 13 Confirmation of Purities of EF-Tu, DsbA, and DsbC

Cells in which proteins comprising EF-Tu with His-Tag were expressed were sonicated in ice so as to be suspended in a loading buffer (300 mM NaCl, 50 mM NaH2PO4, pH 8.0). Then, the resulting cell lysate was subjected to centrifugation (30,000 g, 4° C., 30 minutes). Then, 50% Ni²⁺-NTA slurry (Qiagen), which had been equilibrated in the ice-cooled loading buffer, was added to the supernatant obtained above, followed by stirring at 4° C. for 1 hour. Thereafter, resin was loaded into a column, followed by washing at 4° C. using the loading buffer (containing 10 mM imidazole, pH 8.0) in a volume 20 times that of the column. Using a loading buffer in a volume 20 times that of the column, proteins of interest were eluted from the column with an imidazole concentration gradient of 10 to 250 mM. Then, fractions (1 ml each) were collected. Proteins of interest were confirmed via SDS-PAGE. The electrophoresis pattern of each lane was read using a densitometer such that the purity was calculated. FIG. 13 shows electrophoresis images of EF-Tu elution fractions. The electrophoresis pattern of each lane was read using a densitometer, followed by collection of Ef-Tu fractions having purities of 90% or more.

In addition, FIG. 14 shows electrophoresis images of purified DsbA (lane 1) and DsbC (lane 2). Both enzymes had purities of 90% or more.

INDUSTRIAL APPLICABILITY Protein Synthesis Method

In accordance with the present invention, DTT that preferably has been removed from a conventional reaction solution may be added to a concentration of several micrometers to 1 mM. In addition, it is also possible not to add any DTT.

Further, in accordance with the conventional method using a cell-free protein synthesizing system from which DTT has been removed, it has been generally considered that amounts of synthesized proteins would decrease under the conditions of such system and synthesis efficiency of proteins of interest which have activity would deteriorate even if disulfide bonds could be formed in proteins. However, in accordance with the present invention, with the use of the reconstituted protein synthesizing system, amounts of synthesized proteins remain stable or increase in some cases. Thus, proteins can be synthesized with improved efficiency.

[Enzyme Assay]

In accordance with the method of activity measurement of the present invention, substrates are synthesized as single-strand polypeptides having no disulfide bond as a result of a protein synthesis reaction so that steps of reduction and denaturation are not required. Thus, upon synthesis or after synthesis, enzymes for activity measurement are added such that activities or structures of folded proteins are regarded as indicators. Therefore, the method of the present invention is characterized in that the steps and the time required can be significantly simplified and shortened compared with the conventional method.

All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety. 

The invention claimed is:
 1. A method of synthesizing a protein having intermolecular or intramolecular disulfide bonds, comprising using a reaction system comprising a), b), c), and d) below: a) at least one template nucleic acid encoding a protein of interest, b) a protein synthesis reaction reagent comprising 1) synthesized or isolated ribosomes, initiation factors, elongation factors, termination factors, aminoacyl-tRNA synthetases, methionyl tRNA transformylases, tRNAs, amino acids, ribonucleoside triphospates, 10-formyl 5,6,7,8-tetrahyrofolic acid (FD), salts and water or 2) synthesized or isolated ribosomes, initiation factors, elongation factors, aminoacyl-tRNA synthetases, methionyl tRNA transformylases, tRNAs, amino acids, ribonucleoside triphosphates, 10-formyl 5,6,7,8-tetrahydrofolic acid (FD), salts, and water, c) at least one purified oxidoreductase, and d) an effective amount of a purified dithiothreitol and at least one purified redox reagent, adding c) the at least one purified oxidoreductase for catalyzing oxidation-reduction of disulfide bonds d) the purified dithiothreitol and the at least one redox reagent for conditioning a redox state of disulfide bonds to b) the protein synthesis reaction reagent; thereafter adding the template nucleic acid a) to the protein synthesis reaction reagent b), thereby causing a reaction to synthesize a protein encoded by the template nucleic acid; and allowing the synthesized protein to form intermolecular or intramolecular disulfide bonds.
 2. The method of synthesizing a protein according to claim 1, wherein the oxidoreductase is an enzyme catalyzing promotion and/or isomerization of disulfide bonds of the protein.
 3. The method of synthesizing a protein according to claim 1, wherein the oxidoreductase consists of protein disulfide isomerases (PDIs) or disulfide interchange proteins.
 4. The method of synthesizing a protein according to claim 3, wherein the concentration of protein disulfide isomerase is is 0.001 to 10 μM.
 5. The method of synthesizing a protein according to claim 3, wherein the concentration of disulfide interchange protein is 0.01 to 10 μM.
 6. The method of synthesizing a protein according to claim 1, wherein the reaction system comprises thioredoxin reductase at a concentration in the reaction system which is not more than 100 ng/ml.
 7. The method of synthesizing a protein according to claim 1, wherein the reaction system comprises glutathione reductase at a concentration in the reaction system which is not more than 100 ng/ml.
 8. The method of synthesizing a protein according to claim 1, wherein the redox reagent is oxidized glutathione.
 9. The method of synthesizing a protein according to claim 8, wherein a ratio of a concentration of oxidized glutathione to dithiothreitol (oxidized glutathione:dithiothreitol) is 1:1 to 4:1.
 10. The method of synthesizing a protein according to claim 1, wherein the purified oxidoreductase comprises DsbA and DsbC.
 11. The method of synthesizing a protein according to claim 1, wherein the purified oxidioreductase consists of DsbA and DsbC. 